Samples were resuspended in 100 μL of 100 mM Triethylammonium bicarbonate (TEAB) (Sigma-Aldrich), reduced with 20 mM DTT (Sigma-Aldrich) at room temperature for 60 min and alkylated with 40 mM iodoacetamide (Sigma-Aldrich) at room temperature in the dark for at least 60 min. Samples were digested overnight at 37 ºC with 1 μg of Trypsin (Promega) with the exception of samples for TMT labeling which were digested overnight at 37 ºC with 1 μg Lys-C (Promega). Subsequently, 1 μg of modified Trypsin (Promega) was added, and the samples were incubated for 3-4 h at 37 ºC. Samples were then acidified with TFA (0.1% (v/v) final concentration; Sigma-Aldrich) and centrifuged at 21,000 x g for 10 min, with the supernatant frozen at -80 ºC until required.
For peptide clean-up and quantification, 200 μL of Poros Oligo R3 (Thermo Fisher Scientific) resin slurry (approximately 150-200 μL resin) was packed into Pierce™ Centrifuge Columns (Thermo Fisher Scientific) and equilibrated with 0.1% TFA. Samples were loaded, washed twice with 200 μL 0.1% TFA and eluted with 300 μL 70% acetonitrile (ACN) (adapted from77 (link)). 10 μL was taken from each elution for Qubit™ protein assay (Thermo Fisher Scientific) quantitation, with the remaining sample retained for MS.
For peptide clean-up and quantification, 200 μL of Poros Oligo R3 (Thermo Fisher Scientific) resin slurry (approximately 150-200 μL resin) was packed into Pierce™ Centrifuge Columns (Thermo Fisher Scientific) and equilibrated with 0.1% TFA. Samples were loaded, washed twice with 200 μL 0.1% TFA and eluted with 300 μL 70% acetonitrile (ACN) (adapted from77 (link)). 10 μL was taken from each elution for Qubit™ protein assay (Thermo Fisher Scientific) quantitation, with the remaining sample retained for MS.